Preparation useful for, and method for treatment of neonatal alloimmune thrombocytopenia (NAIT)

ABSTRACT

A preparation useful for, and a method for the prophylactic treatment of women post-childbirth in order to avoid immunization and antibody production, which could induce NAIT and fetal/neonatal bleeding in subsequent pregnancies comprising administering a preparation containing antibodies to HPA1a within 72 hours after delivery in the first non-compatible pregnancy.

BACKGROUND

1. Field of the Invention

The invention relates to a preparation useful for, and a method for theprophylactic treatment of women post-childbirth in order to avoidimmunization and antibody production, which could induce NAIT andfetal/neonatal bleeding in subsequent pregnancies.

2. Background of the Invention

Neonatal Alloimmune Thrombocytopenic Purpura. (NAITP)

Two percent of Caucasians are homozygous for human platelet antigen(HPA) 1b. The HPA 1a antigen is a potent immunogen and ten percent ofpregnant HPA 1a negative women make antibodies to the HPA 1a antigenafter immunization with their fetus' HPA 1a positive platelets. Most ofthese women have the major histocompatibility antigen HLA-DRB3*0101, butthere are examples of women with other HLA DR antigens making theantibodies. The immunization can take place early in the first pregnancymaking the fetus thrombocytopenic as early as the 16-20^(th) week ofgestation. Intracranial hemorrhage may be fatal, or the fetus cansurvive with neurological sequels. Fetal alloimmune thrombocytopenia isreported to be present in 1:1000-2000 pregnancies.

Most of the studies reporting frequencies of anti-HPA 1a antibodies havebeen done retrospectively in women giving birth to thrombocytopenicbabies with symptoms of impaired hemostasis. Recently a prospectivestudy of 100 448 pregnant women showed a frequency of HPA 1bb of 2.1%.10,6% of the women at risk had anti-HPA 1a antibodies and 55 babies hadsevere thrombocytopenia.

At present there is no general agreement about how to manage thefollow-up of the pregnant women with anti-HPA 1a antibodies in order toreduce the risk for bleeding in the fetus/newborn. There is no reliableprenatal parameter to predict which fetuses that are susceptible tolife-threatening thrombocytopenia and therefore need closer follow-up orintervention.

In order to approach the questions related to predictable tests forthrombocytopenia and management of the babies to reduce the risk ofbleeding, we undertook a prospective investigation of samples frompregnant women referred to our laboratory at the Departments forimmunology and transfusion medicine, University Hospital og NorthernNorway and Ullev{dot over (a)}l University Hospital, for Rhesus D (RhD)testing.

Until now, the general opinion was that immunization with HPA 1a antigentook place during the first non-compatible pregnancy. Our research hasdisclosed, however, that in 70-80% of those women with antibodies to HPA1a, immunization occurs in association with delivery, as antibodies canbe detected 6 weeks post partum but not at the time of delivery. This isa very interesting observation, and shows that the time for immunizationin NAIT is very similar to that seen in haemolytic disease of thenewborn (HDN), contrary to the currently held belief in the art.

Hemolytic Disease of the Newborn (HDN).

A Rhesus D (RhD) negative woman with an RhD positive foetus, can makeantibodies against the erythrocytes of her child if red cells enter hercirculation. Her antibodies of the IgG class can transfer the placentalbarrier and destroy the red cells of the foetus. Hemolysis and anemiaare the most common results of such antibody transfer, but the mostfeared complications are hydrops foetalis and death. In HDN, theimmunization takes place when the first child is born, and antibodies toRh(D) can be detected after termination of the pregnancy. Antibodies arenot a problem in the first pregnancy, but may affect the nextnon-compatible child.

Today it is possible to prevent the generation of anti-Rh(D) antibodiesin association with pregnancy. Within 72 hours after delivery, the womanis given an intramuscular injection of antibodies to the antigen, namelyanti-Rh(D). The accepted explanation for the effect is that suchantibodies will destroy or remove the fetal red cells that have passedinto the circulation of the mother, and prevent immunization and theformation of antibodies to the Rh(D) antigen.

The antibody preparation is an IgG concentrate made from the plasma ofindividuals with anti-Rh(D) in their circulation. Normal individuals mayhave anti-Rh(D) as a consequence of insufficient prophylaxis withanti-Rh(D) in D-negative pregnant women, transfusion of Rh(D) positiveblood to Rh(D) negative recipients, or as a result of activeimmunizations. “Rhesogamma P<<ZLB Behring>>” is a human immunoglobulincontaining anti-D for prevention of HDN. 1.5 ml of the preparationcontains 1500 IE (200 microgram) of anti-D, which makes up onetherapeutic dose. The total amount of IgG in one dose is 255 mg. Tendoses are sold for the price of 3200 NOK.

A prerequisite for an efficient treatment is that the prophylaxis isgiven before the immune response is established in the mother. This isthe case in HDN, as the immunization and antibody production takes placesubsequent to the delivery of the first child. The treatment is veryefficient, and HDN due to anti-D is seldom seen today.

Current Treatment of Nait

At present, there is no prophylactic treatment for NAIT as is the casewith HDN. Newborns with NAIT are treated with platelet transfusions orintravenous injections of gamma globulins after birth. If a womandelivers a child with severe NAIT, she may herself be treated with highdose intravenous IgG and/or steroids in her next pregnancy. Inparticular cases, the child may be transfused with compatible plateletsseveral times during the second half of the pregnancy. This procedure isassociated with a high mortality rate, about 1% in each puncture. Thesetreatment modalities are only eligible when the woman has given birth toa thrombocytopenic child in a previous pregnancy. So, the first child isborn without any kind of precautionary action. Other postnatal treatmentmay anyhow come too late, as the damage to the child may occur duringdelivery or shortly after.

SUMMARY OF THE INVENTION

The current opinion in this field is that the immunization of the motheroccurs during, and not after the first non-compatible pregnancy.Therefore, prophylactic treatment has not been an option. Now, with oursurprising observation that immunization, in about 70-80% of cases,takes place after the first non-compatible pregnancy, there is goodreason to believe that also this condition could be influenced by asimilar treatment as is used for HDN.

Rh(D) negative women giving birth to Rh(D) positive children, have therisk to produce antibodies to the Rh(D) antigen present on the childserythrocytes. The stimulation to such antibody production, is a resultof fetal erythrocytes entering the mothers circulation in associationwith delivery. The crucial point is that the mother has not beenstimulated with fetal erythrocytes during the pregnancy, so that thefirst stimulus she receives, is that resulting from fetal erythrocytesat delivery. If the mother is given antibodies to Rh(D) after she hasreceived the Rh(D) positive fetal erythrocytes into her circulation, butbefore her immune system starts to produce her own antibodies to Rh(D),this production is blocked. It is believed that the injected antibodiesbind to the Rh(D) positive fetal erythrocytes and destroy them beforethey are able to stimulate the mothers immune system to antibodyproduction.

According to one aspect of the invention, we can substitute Rh(D),erythrocytes and antibodies to Rh(D), with HPA 1a, platelets andantibodies to HPA 1a, respectively.

HPA 1a negative women giving birth to HPA 1a positive children, have therisk to produce antibodies to the HPA 1a antigen present on the child'splatelets. The stimulation to such antibody production, is a result offetal platelets entering the mothers circulation in association withdelivery. The crucial point is that the mother has not been stimulatedwith fetal platelets during the pregnancy, so that the first stimulusshe receives, is that resulting from fetal platelets at delivery. If themother is given antibodies to HPA 1a after she has received the HPA 1apositive fetal platelets into her circulation, but before her immunesystem starts to produce her own antibodies to HPA 1a, this productionwill be blocked. It is believed that the injected antibodies will bindto the HPA 1a positive fetal platelets and destroy them before they areable to stimulate the mothers immune system to antibody production.

Accordingly, the current invention provides for a prophylactic regimesimilar to that for HDN but applied for the prophylactic treatment ofNAIT.

An immunoglobulin fraction containing antibodies to HPA 1a can beisolated from individuals with high levels of the antibody. Women whohave such antibodies as a result of previous incompatible pregnancies,would be the most preferred donors. The preparation is made by isolationof total IgG from human plasma containing anti-HPA 1a. A therapeuticdose of 0.5-2.0 ml, preferably 1.5 ml containing 250-300 mg IgG, isadministered by injection as soon as possible after delivery, and atlatest within 72 hours. The injection is given once.

DETAILED DESCRIPTION OF THE INVENTION

Based upon the unpublished research of the inventors described in U.S.provisional patent application 60/755,062 filed 3 Jan. 2006, the entirecontents of which are incorporated by reference as if fully repeatedherein, it has been surprisingly discovered that in 70-80% of pregnantwomen with antibodies to HPA 1a, immunization occurs in association withdelivery as opposed to during the first non-compatible pregnancy. Thisconclusion is based upon the observation of subjects where antibodiescan be detected 6 weeks post partum but not at the time of delivery.This is a very interesting observation, and shows that the time forimmunization in NAIT is very similar to that seen in haemolytic diseaseof the newborn (HDN), contrary to the currently held belief in the art.Based upon this correlation with HDN, the present invention provides apreparation useful for, and a method of treatment of NAIT that similarto that for HDN.

The Preparation

It is current practice today to fractionate IgG from normal blood donorplasma for the treatment of immunodeficiency. Plasma is collected in theblood banks, and fractionation is performed by a collaborating company.The immunoglobulin preparation is accepted for use by StatensLegemiddelverk (The Norwegian Government body regulatingpharmaceuticals).

For the purpose of the present invention it is preferable to selectdonors with high levels of anti-HPA 1a. In the context of the invention,donors with “high levels” are preferably women who have given birth tochildren with NAIT, who in 80-90% of cases, have anti-HPA 1a levelsabove 200 AU/ml. To define arbitrary units (AU), we selected a serumfrom a woman who had a child with severe thrombocytopenia. Her serum wasgiven the quantity of anti-HPA 1a of 1000 AU/ml. This serum is used tocreate a standard curve for quantitation of other women's antibodylevels. According to the invention, we select sera from women with “highlevels” (>1000 AU/ml) for production of IgG for the prophylacticpreparation.

Based upon the above criteria, enough plasma is collected to make up aprocessable batch. The batch is thereafter fractionated by methods knownin the fractionation industry. Such methods include isolatingImmunoglobulin G from plasma by ion exchange chromatography orimmunoadsorption techniques or by adsorption to protein A-Sepharose.

1 liter of plasma yields about 4.0 g IgG, and 1 therapeutic dose ofanti-D consists of about 255 mg IgG. If we assume that the same amountof IgG from anti-HPA 1a plasma would be sufficient for 1 therapeuticdose, it means that 1 liter of plasma will give 16 therapeutic dosescontaining from about 100-400, preferably from 200-300 mg IgG.

In order to make sure that the preparation contains antibodies to HPA 1ain sufficient amounts, we will perform neutralization experiments withintact platelets. Comparison with the anti-D preparation that is usedfor HDN prophylaxis will give an indication of the potential of thegiven anti-HPA 1 a preparation. Preparations of 10 with either anti-Rh(D) or anti-HPA 1a at a certain IgG concentration (250 mg/ml) is mixedwith defined amounts of erythrocytes or platelets with the correspondingantigens on the surface. By mixing different amounts of platelets anderythrocytes to the antibody preparations, it can be determined exactlyhow much platelets or erythrocytes that is required to neutralize therespective antibodies in the two different IgG preparations. Plateletsand erythrocytes have defined amounts (numbers) of antigens on thesurface; therefore it can be determined how many antibodies of therespective specificities that are present in each preparation. ByadjUstments of the IgG concentration in each preparation, equimolecularsolutions with regard to antibody molecules can be made. In this way weintend to make a preparation of anti-HPA 1a that contains as manyspecific antibody molecules as the anti-Rhesus(D) preparation. Thehypothesis is that the same number of antibody molecules has the samepotential to block the immune response.

An advantage of a the preparation made by the above-describe method isthat it will be easily approved for use, and will not need to go throughthe whole time consuming registration process that is mandatory for theintroduction of new drugs.

Product Characterization:

The preparation according to the invention comprises a concentrate madeby the isolation of total IgG from human plasma. The preparationcontains representative amounts of all antibody specificities that arepresent in the actual sera collected for processing. In addition tothis, the preparation contains IgG antibodies specific for HPA 1a atsufficient levels to inhibit an immune response in a subject to HPA 1aantigens. Those antibodies will comprise an amount of the total IgG offrom about 0.1% to 2% by weight. The antibodies are dissolved in salinecontaining preservatives.

Therapeutic Dose:

A therapeutic dose is between 0.5 ml-2.0 ml of the preparation,preferably 1.5 ml. 1.5 ml of the preparation contains specificantibodies to HPA 1a. One therapeutic dose contains over 200 mg IgG,preferably from 250-300 mg IgG.

Prevention of Immunization with HPA 1a Positive Platelets

The target individuals for the injections are women, immediately afterdelivery in their first pregnancy. If they have been exposed to theantigen at an earlier occasion, the effect of the treatment could beminimal.

To trace the target individuals, one aspect of the invention providesfor a screening procedure. If 50,000 pregnants are genotyped for the HPA1 antigen, we will find about 1,000 HPA 1a negative women. About 400 ofthose are primigravidae, and it is expected that about 40 of them willmake antibodies to HPA 1a. 10 will make antibodies during the pregnancy,and 30 after delivery.

The object for the treatment according to the invention is to reduce thenumber of women that make antibodies after delivery. The women who haveantibodies at time of delivery will not be treated. For the 30 who makeantibodies after delivery, we expect to block the immune response in 90%of the cases.

Administration

One therapeutic dose of the preparation according to the invention isinjected intramuscularly as soon as possible, and within 72 hours afterdelivery.

Frequency of Injection.

The injection is given once.

Frequency of Treatments in Norway Per Year

About 70,000 pregnancies are initiated each year in Norway. This figureincludes normal deliveries, spontaneous and provoked abortions. If 2.1%of all Caucasians are HPA 1a negative, this means that about 1,500pregnants have to be treated each year. Another possible indication forprophylaxis is HPA 1a negative women at fertile age that are transfusedwith HPA 1a positive blood. This would be between 200 and 400 patientseach year.

EXAMPLE Confirmation of Effectiveness of Treatment

Antibodies 6 Weeks After Delivery:

About 1,000 HPA 1a negative women will be identified. About 400 of thoseare primigravidae, and it is expected that about 40 of them will makeantibodies to HPA 1a. 10 will make antibodies during the pregnancy, and30 after delivery. Our goal is to reduce the number of women that makeantibodies after delivery. Those women who have antibodies at time ofdelivery will not be treated. The 370 primigravidae with no detectableanti-HPA 1a at delivery will be treated. Antibody levels will beanalysed 6 weeks after delivery. For the 30 who normally are expected tomake antibodies 6 weeks after delivery, we do not expect to findanti-HPA 1a antibodies in 70-90% of them after treatment.

The invention claimed is:
 1. A method for the prophylactic treatment ofneonatal alloimmune thrombocytopenia (NAIT), the method comprising thestep of administering to a pregnant woman who is negative for the humanplatelet antigen HPA 1a, as well as negative for antibodies specific tothe human platelet antigen HPA 1a, and who has received HPA 1a positivefoetal platelets into her circulation from her HPA 1a positive foetus, apreparation comprising a sufficient quantity of antibodies to HPA 1a soas to substantially inhibit the woman's immune system from producingantibodies to HPA 1a wherein the preparation is administered to thewoman before her own immune system starts to produce her own antibodiesto the HPA 1a of the foetal platelets.
 2. The method of claim 1, whereinthe preparation is administered within 72 hours of receiving HPA 1apositive foetal platelets into her circulation.
 3. The method of claim2, wherein the preparation comprises Immunoglobulin G plasma derivedfrom human plasma.
 4. The method of claim 2, wherein the preparationthat is administered comprises an immunoglobulin G (IgG) concentratederived from human plasma containing antibodies specific to humanplatelet antigen HPA 1a.
 5. The method of claim 4, comprising thefurther step of determining whether the newborn is positive for humanplatelet antigen HPA 1a prior to administering the preparation to thewoman.
 6. A method for the prophylactic treatment of neonatal alloimmunethrombocytopenia (NAIT), the method comprising the step of:administering to a woman who is negative for the human platelet antigenHPA 1a, as well as negative for antibodies specific to the humanplatelet antigen HPA 1a, and who has received or who may receive HPA 1apositive foetal platelets into her circulation at delivery of her HPA 1apositive child, a preparation comprising a sufficient quantity ofantibodies to HPA 1a so as to substantially inhibit the woman's immunesystem from producing antibodies to HPA 1a, wherein the preparation isadministered to the woman within 72 hours of delivery.
 7. The method ofclaim 6, wherein the preparation is administered up to 72 hours afterdelivery.
 8. The method of claim 6, wherein the preparation isadministered up to 72 hours before delivery.
 9. The method of claim 6,wherein the preparation comprises Immunoglobulin G plasma derived fromhuman plasma.
 10. The method of claim 6, wherein the preparation that isadministered comprises an Immunoglobulin G (IgG) concentrate derivedfrom human plasma containing antibodies specific to human plateletantigen HPA 1a.
 11. The method of claim 6, comprising the further stepof determining whether the newborn is positive for human plateletantigen HPA 1a prior to administering the preparation to the woman.